Nogo-A调控阿尔茨海默病Aβ代谢的分子机制

The Nogo-A is known to contribute to inhibiting the neurite regeneration of neurons after traumatic injuries. Recent evidences suggest that Nogo-A is involved in the pathology of Alzheimer's disease(AD) and there remains a need to determine it's exact effect and underlying mechanism.Our initial study found that the active fragment of the Nogo-A inhibited neurite outgrowth and increased amyloid β-protein(Aβ)secretion of cortical neurons by activating Nogo-66 receptor(NgR).Based on our research work,this study will further explore the mechanisms of Nogo-A regulates the metabolism of Aβ by using Western blot, RNA interference, co-immmunoprecipitation technology et al, focus on: 1)NgR activated signal trnasduction process, explolre the pathway of the downstream signaling molecules Rho-associated coiled coil-containing protein kinase（ROCK）and protein kinase C regulate key enzymes(secretase,neprilysin etc) of Aβ metabolism; 2)The competitive inhibition pathway of Aβ generation, Nogo-A inhibits the combination of amyloid β-protein precursor (APP) and NgR by bonding to NgR competitively,promotes the hydrolysis of APP which results in increased Aβ generation; 3) To study the effects of Nogo-A on Aβ metabolism, pathological and behavioral changes of normal and AD model mice and confirm whether the mechanisms are consistent with the above mechanisms.This study will provide new opinins and targets on the regulation of Aβ metabolism, and experimental evidences for the role of Nogo-A in the pathology of AD.

近年研究表明，外伤后抑制神经再生的Nogo-A与阿尔茨海默病（AD）的发病有联系，其作用仍不清楚。我们前期研究发现，Nogo-A上的活性片段激活Nogo-66受体(NgR),抑制皮质神经元突起生长并增加β淀粉样蛋白(Aβ)的生成。本研究拟采用Western blot、RNA干扰、免疫共沉淀等技术，进一步深入研究Nogo-A调控Aβ代谢的分子机制，重点解决：1)NgR激活后介导的信号途径。阐明其下游信号分子ROCK和蛋白激酶C对Aβ代谢的关键酶(分泌酶、脑啡肽酶等)的调节通路；2）竞争抑制途径。明确Nogo-A是否通过竞争抑制淀粉样蛋白前体(APP)与NgR的结合,促进APP水解而增加Aβ生成；3)Nogo-A对正常和AD模型鼠脑内 Aβ代谢、病理和行为学的影响，是否通过以上机制起作用。本研究将拓展Aβ代谢的调控途径及干预其过度生成提供新靶点，也为Nogo-A在AD发病中的作用提供实验依据。

近年研究表明，抑制神经再生的Nogo-A与阿尔茨海默病发病有联系，但其作用仍不清楚。本研究旨在研究Nogo-A对β淀粉样蛋白(Aβ)代谢的调控作用及机制，阐明其调控的具体信号通路。.研究内容主要包括两部分。首先是表达纯化Nogo-A的活性功能片段Nogo-66。由于目前无商品化的Nogo-66蛋白，我们利用大肠杆菌表达系统可溶性表达SUMO-Nogo-66融合蛋白，通过SUMO蛋白酶切割及亲和层析方法获得纯度大于95%的重组Nogo-66蛋白，终产量达到23±1.5mg/L。蛋白质肽谱和测序结果显示，重组Nogo-66蛋白与天然蛋白序列一致（此方法可有效解决Nogo-66蛋白在原核表达系统中遇到的包涵体表达及活性低问题）。在以上基础上研究Nogo-A调控Aβ代谢的分子机制。研究发现，Nogo-A蛋白活性功能片段Nogo-66能增加皮质神经元Aβ的生成，Aβ42和Aβ40的产量同时增加，其调控机制主要由两条信号通路来完成。1）Nogo-66与Nogo-A受体（NgR）结合后，引起NgR下游信号分子ROCK的激活，激活的ROCK对α分泌酶活性和含量无影响，也不影响β分泌酶的含量，但能增加β分泌酶的活性，C99的产量增加，使得β淀粉样蛋白前体(APP)主要通过β分泌酶途径代谢，Aβ的生成增加；2）Nogo-66与NgR结合激活ROCK后，还能增加APP蛋白的表达，从而增加Aβ的生成。.本研究发现Nogo-A除了神经再生抑制功能外，还能调控APP代谢，增加Aβ42和Aβ40的生成，初步阐明了Nogo-A调控Aβ代谢的分子机制，获得了两条完整的信号调控通路。